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KAPA HTP DNA LIBRARY PREPARATION FOR ILLUMINA SEQUENCING ON THESCICLONE NGS WORKSTATION

Application: Maestro KAPA DNA Library Preparation Rev1

Compatible with: KAPA HTP Library Preparation Kit for Illumina® Platforms, v3.12 Sciclone® NGS Workstation with Maestro™ 6.0 Sciclone NGSx Workstation with Maestro 6.0 or 6.3

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Introduction 3

Maestro KAPA Library Prep Workflow Overview 3

Required Materials and Reagents 5

Reagents 5

General Lab Equipment and Supplies 5

Sciclone NGS Workstation Accessories 5

Consumables 6

Running the Maestro KAPA Library Prep Application 6

Sample Preparation 6

KAPA Library Prep Run Preparation Steps 6

PCR Enrichment 8

KAPA Post-PCR SPRI Run Preparation Steps 9

Library QC and storage 10

Expected Results 10

Appendix A: Step-by-Step Guide to the KAPA Library Prep Application 11

Appendix B: Step-by-Step Guide to the KAPA Post-PCR SPRI Application 12

Table of Contents

Maestro KAPA DNA Library Preparation Rev1

Two independent Maestro Applications are used in the 1-day KAPA Library Prep Workflow.

The Maestro KAPA sample preparation closely follows the protocol recommended by KAPA Biosystems. A single aliquot of AMPure XP® beads is used per sample for the post-End Repair, post-A-tailing and post-Ligation SPRI steps. This strategy increases yield by limiting the number of times samples are transferred to new wells/plates and decreases the cost of the assay by reducing AMPure XP® bead consumption. For SPRI cleanup steps, the DNA in the sample is driven onto the beads via addition of PEG/NaCl SPRI solution. For the A-tailing and Ligation steps, DNA is eluted from the beads directly into the reaction mixture. In the Maestro KAPA Library Prep application, the amount of water in the A-tailing and Ligation master mixes is reduced, and the reaction is assembled in two steps. First, 30 µL water is dispensed into the wells with DNA/beads. Second, 20 µL of master mix is dispensed into the wells and the DNA/beads are resuspended in 50 µL total reaction volume. Adding water to the beads separately allows the reaction master mixes to be pre-arrayed by the Sciclone NGS workstation into a 384-well staging plate. Use of the 384-well staging plate reduces the consumables needed in the workflow, while ensuring that reaction mixes are added simultaneously to all wells of the sample plate.

Three different workflow options are available for post-ligation cleanup of samples prior to setup for PCR enrichment of the libraries. The “Single SPRI” option performs a single “0.5X” cleanup step before PCR setup. After the ligation reaction incubation, 25 µL of PEG/NaCl SPRI solution are added to each 50 µL ligation reaction. After the SPRI cleanup, DNA is eluted in 22 µL 10 mM Tris pH 8.0 and 20 µL is transferred into the PCR reaction. The “Double SPRI” option performs two consecutive, “1X” post-ligation SPRI cleanups. After the ligation reaction incubation, the first post-ligation SPRI cleanup as described above, except that 50 µL of PEG/NaCl SPRI solution is added to each 50 µL ligation reaction, and elution is in 50 µL 10 mM Tris pH 8.0. To the 50 µL eluted sample, 50 µL PEG/NaCl SPRI solution is added for a second SPRI cleanup and elution is done in 22 µL 10 mM Tris pH 8.0. The “Size Selection” option performs a dual “0.55X – 0.75X” SPRI size selection. The initial “1X” post-ligation SPRI cleanup is performed, with elution in 100 µL 10 mM Tris pH 8.0. To the 100 µL sample, 55 µL PEG/NaCl SPRI solution is added to bind fragments larger than ~450 bp. After

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Introduction

Preparation of DNA samples for cluster generation and sequencing on the Illumina® platform requires a series of manipulations to efficiently ligate appropriate indexed adapters onto DNA fragments to produce paired-end libraries. Automating the process has the advantage of avoiding sample tracking errors and reducing sample-to-sample variability while dramatically increasing throughput. The Maestro-based KAPA Library Prep Workflow from PerkinElmer provides a pre-programmed solution for sample preparation on the Sciclone NGS workstation for KAPA Biosystems’ KAPA HTP Library Preparation Kit v3.12.

Figure 1. Overview of the steps for KAPA library preparation on the Sciclone NGS Workstation

Set-up Run Time (including Maestro Application Time thermocycler steps)

KAPA Library Prep 1 hour 5-6 hours

KAPA Post-PCR SPRI 10 min 45 min

Maestro KAPA Library Prep Workflow Overview

The Maestro Application for KAPA library preparation is a validated process for library preparation from fragmented DNA samples that follows the steps outlined in Figure 1. Samples are processed in 96-well PCR plates. The number of samples to process (1 to 12 columns of 8 samples each) is selected at the start of each run. Tip-tracking utilities guide partial tip box loading and ensure that each tip box is used to completion prior to starting the next box. Inheco temperature blocks installed on the Sciclone NGS Workstation deck allow for appropriate 4 °C storage of reagents and heated incubations of reactions. Reaction mixes are pre-arrayed prior to addition to sample to ensure equal incubation times across the sample plate. Adapter indexing patterns may be executed during the run by arraying 1 - 24 different adapters to appropriate well locations as specified by the user in an Excel workbook. Easy-to-follow user interfaces guide the reagent and deck setup process and prompt the user for any necessary interventions.

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binding, the supernatant is transferred from the beads to a plate containing 20 µL fresh.AMPure XP® reagent. The concentration of PEG/NaCl in this second plate favors binding of fragments >200 bp in size. After washing the beads with ethanol and drying, the 250 - 450 bp fragments are eluted into 22 µL 10 mM Tris pH 8.0 and 20 µL is transferred to the PCR reaction.

During startup of the KAPA Library Prep application, the user will be prompted to select the desired workflow in the window shown in Figure 2. The appropriate choice for post-ligation cleanup depends on the user’s workflow and the intended downstream processing of amplified library samples. Please consult the KAPA Biosystems Technical Data Sheet for the KAPA HTP Library Preparation Kit for guidance on adapter concentrations, PCR cycling parameters and post-ligation cleanup options.

Figure 2. Selection of Post-ligation SPRI cleanup option.

Figure 3. KAPA Library Prep application steps

Figure 4. KAPA Post-PCR SPRI application steps

Broadcast 22 µL EndRepair Mix to 96-well plate

Transfer 20 µL EndRepair Mix to 50 µL sample

Incubate 30 min at 20C; Broadcast A-Tail mix and Ligase mix to 384-plate

SPRI cleanup; 120 µL beads, elute in 30 µL water,

leave on beads

Transfer 20 µL A-Tail Mix to sample and mix

Incubate 30 min at 37C; Broadcast Adapters to

384-well plate

SPRI cleanup, 90 µL PEG/NaCl, elute in 25 µL

water, leave on beads

Transfer 20 µL Ligase Mix and 5 µL adapter to sample

Incubate 15 min at 20C


(Optional: repeat SPRI cleanup or Dual SPRI size

selection. Elute in 22 µL EB)

Broadcast 30 µL; PCR MM to PCR plate

Transfer 20 µL sample to PCR plate

Amplify on Thermocycler

Broadcast 22 µL EndRepair Mix to 96-well plate

Transfer 20 µL EndRepair Mix to 50 µL sample

Incubate 30 min at 20C; Broadcast A-Tail mix and Ligase mix to 384-plate

SPRI cleanup; 120 µL beads, elute in 30 µL water,

leave on beads

Transfer 20 µL A-Tail Mix to sample and mix

Incubate 30 min at 37C; Broadcast Adapters to

384-well plate


water, leave on beads

Transfer 20 µL Ligase Mix and 5 µL adapter to sample

Incubate 15 min at 20C


(Optional: repeat SPRI cleanup or Dual SPRI size

selection. Elute in 22 µL EB)

Broadcast 30 µL; PCR MM to PCR plate

Transfer 20 µL sample to PCR plate

Amplify on Thermocycler

Transfer 50 µL PCR reaction to clean plate

Add 50 µL AMPure® beads; SPRI cleanup, elute in 32 µL

Transfer 30 µL library to clean plate

QC and store at -20C

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Required Materials and Reagents

Reagents

Reagent Vendor and Part No.

KAPA HTP Library Preparation Kit v3.12 for Illumina® Platforms (96 samples) KAPA Biosystems KK8234

Indexed Y-adapters of the Illumina® TruSeq™ design Various

PCR primers of the Illumina® TruSeq™ design Various

Nuclease-Free Water Various

Ampure XP® reagent Beckman Coulter-- A63881 (60 mL)

100% EtOH Sigma E7023

10 mM Tris pH 8.0 Various

Note: KAPA HTP kits supply enough reagents to process 96 samples. While overage volumes are generous, if kits are used in numerous runs with < 96 samples per run volumes of specific reagents may become limiting. Take care to store and handle KAPA reagents as specified and avoid multiple freeze-thaw cycles.

Note: Adapters and PCR primers are not supplied with the KAPA HTP Library Preparation Kit, and must be obtained separately.

General Laboratory Equipment and Supplies

Equipment Supplier

Microfuge Various

Vortexer Various

200 µL Multichannel pipettor and appropriate barrier tips Various

1000 µL Multichannel pipettor and appropriate barrier tips Various

Covaris™ S2 or E210 System and appropriate tubes Covaris™

Microplate centrifuge Various

Thermocycler** BioRad (MJ Research) DNA Engine PTC-200, or equivalent

Plate Seals Various- compatible with Thermocycler and freezer storage

LabChip® GX or Agilent Bioanalyzer 2100 with appropriate chips and reagents Various

**The standard sample plates used in this application are fully skirted Bio-Rad Hard-Shell® 96-PCR plates. Please check if your thermocycler is compatible with this plate type. If it is not, please contact your PerkinElmer Field Application Scientist to discuss modifications to the application to support semi-skirted PCR plates.

Sciclone NGS Workstation Accessories

Accessory Part No.

Agencourt® 96-ring magnet CLS 128316

Inheco 384-well plate adapter CLS 100853

Inheco 96-well adapters (2) CLS 128372

Inheco 96-well adapter/shaker CLS 100852

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The sample number must be set by indicating the number of columns to run in the worksheet titled “KAPA Library Prep”. The Maestro application only processes full columns of 8 samples each.

The adapter index pattern is set by designating the appropriate adapter numbers to each well ID in spreadsheet titled “Adapter_Indexing”. Up to 32 different adapters may be used, but the default setting for the spreadsheet will show the 24 index numbers used in the Illumina® TruSeq™ design. TruSeq™ adapters are numbered from ID001 to ID027, with adapters ID017, ID024 and ID026 omitted. If desired, alternative names may be entered in column I.

Enter a number in column B indicating which of the indexes is to be used for each of the samples to be run. The chart on the right will automatically update the number of samples using each adapter index. This information will be passed into the KAPA Library Prep worksheet to indicate the appropriate well and the volume necessary for each adapter index mix.

Save the modified spreadsheet with its original name in its original file path. Print the “KAPA Library Prep” spreadsheet to use as a guide while setting up reagents.


Consumables

PerkinElmer Vendor and No. Used Consumable Description Part No. Part No. per Run

PCR Plates* 96-well PCR Plate, Bio-Rad Hard-Shell®, Full Skirt 6008870 Bio-Rad HSP-9631 10-12

Boxed Tips* Pipette Tip-150 µL, Art, Box, 10-96 Sterile Racks 111426 20-26 (96 samples)

Deepwell Plates Deepwell-96 POS, Square 2.0 mL well, Polypro, Seahorse 6008880 Seahorse Bioscience 201379-100 2

Deepwell Reservoirs Reservoir-Deepwell, 12-col Pyramid Bottom, 290 mL, Seahorse 6008730 Seahorse Bioscience 201250-100 2

Lids 946 Lid-Universal, Robotic Friendly, Polystyrene 6000030 Seahorse Bioscience 200856-100 6

384-well Plate Microplate-384-well, Round Bottom, Polypropylene, Pkg of 10 6008890 Corning, Inc. 3672 1

*Plate and tip usage will depend on selected post-ligation cleanup option.

Figure 5. “KAPA Library Prep” worksheet.

Running the Maestro KAPA Library Prep Workflow

Please read and familiarize yourself with all steps described in this section prior to beginning the run. For best results, the entire process should be completed in one day. Allow 6 - 7 hours to complete all steps, including reagent distribution, deck setup, end-repair, A-tailing, ligation, PCR enrichment, and post-PCR cleanup of samples.

Sample preparation

Genomic DNA should be fragmented to an average size of 300 - 400 bp on a Covaris S2 or E210 instrument. Samples should be presented for the Maestro KAPA Library Prep run as 100 ng to 5 µg sheared DNA in 50 µL 10 mM Tris pH 8.0, low TE, or water in a BioRad Hardshell® 96-well PCR plate.

KAPA Library Prep Run Preparation Steps

Note: AMPureXP® beads and PEG/NaCl SPRI solution be equilibrated to room temperature for about 30 minutes before use. They may be taken out of 4 °C storage before beginning. Do not thaw KAPA reagents until steps 1 and 2 have been completed.

If necessary, boot up the system by first starting the Sciclone and the Inheco units, then starting the PC controller.

1. Modify the Maestro KAPA Library Prep Workbook to specify the number of samples to run and the adapter index pattern.

The workbook must be located in the filepath: C:\ProgramData\Caliperls\Maestro\Workbooks\ and must have the name “KAPA Library Prep Workbook.xls”. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

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Figure 6. “Adapter_Indexing” spreadsheet in the “KAPA Library Prep” Workbook.

2. Start the Maestro KAPA Library Prep Application

Launch he Maestro software and open the KAPA Library Prep Application. Start the run by selecting the play button. If running in Editor Mode, be sure to start the Main Method.

Note: When the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps. Starting the application prior to thawing and diluting reagents ensures that the cold blocks are pre-chilled and ready for on-deck reagent storage.

Ensure that the Inheco units have the correct adapters for the run:

Position A3 96-well PCR Plate Adapter Position A4 384-well Plate Adapter Position D2 96-well PCR Plate Adapter Position D4 96-well PCR Plate Shaker Adapter

Verify that the Inheco units for positions A3, A4 are set to 4 °C and are cooling.

3. Prepare the 80% EtOH and Water Reservoirs

Make 100 mL fresh 80% EtOH solution by diluting 80 mL 100% Ethanol with 20 mL nuclease-free molecular biology grade water. Pour 100 mL fresh 80% EtOH into a Seahorse deepwell reservoir, cover with a lid and store at room temperature.

Prepare a second Seahorse deepwell reservoir with 50 mL Nuclease-free water.

4. Prepare PEG/NaCl, 10 mM Tris pH 8.0, and AMPure® XP bead plates

Use the KAPA Library Prep spreadsheet as a guide for setting up the plates.

Using a multichannel pipettor, aliquot the appropriate volume of PEG/NaCl solution per well into a BioRad Hardshell® PCR plate for each column of samples to be run. The volume of PEG/NaCl solution will differ depending on which post-ligation SPRI option has been chosen for the workflow. Label the plate, cover with a lid, and store at room temperature.

Using a multichannel pipettor, aliquot 162 µL 10 mM Tris pH 8.0 per well into a BioRad Hardshell® PCR plate for each column of samples to be run. Label the plate, cover with a lid, and store at room temperature.

Thoroughly resuspend AMPureXP® beads by inverting or rotating the AMPure XP® reagent container. Using a multichannel pipettor, aliquot 125 µL AMPureXP® beads per well into a BioRad Hardshell® PCR plate for each column of samples to be run. Cover the plate with a lid and store at room temperature.

If the “Size Selection” workflow is to be run, additional plates containing 20 µL AMPure XP® reagent (resuspended beads) and 70 µL PEG/NaCl solution will be needed at the start of Step 5. These plates may be prepared at this time or after the Sciclone NGS Workstation run has been started.

Inspect all plates to ensure that air has not been trapped in the wells. If necessary, spin the plates briefly to bring reagents to the bottom of the wells.

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5. Make the reaction master mixes and adapter mixes

The following mixes should be prepared according to the recipes in the KAPA Library Prep spreadsheet: End-Repair Mix, A-Tailing Mix, Ligation Mix, and PCR Mix.

Thaw the KAPA reagents stored at -20 °C on ice. Ensure that each KAPA supplied component has been fully thawed, gently mixed, and spun down before use. After adding the appropriate volumes of reagents in nuclease-free tubes, gently mix and spin again to collect all liquid at the bottoms of the tubes.

Care should be taken to pipet accurately as minimal overage volumes are used. Keep the reaction mixes on ice. Promptly return unused portions of reagents to -20 °C storage.

6. Aliquot the reaction mixes and adapters into plates

Use the KAPA Library Prep spreadsheet as a guide for setting up the Master Mix plate.

Aliquot the appropriate volume of each indexed adapter in the indicated well in column 9 - 12.

Aliquot the specified volumes of End Repair Mix, A-tailing Mix, Ligase Mix, and PCR Mix to columns 1 through 8. Keep the plate on ice while pipetting to keep the reagents cold. Pipet carefully into the bottom of the wells and avoid trapping air or creating bubbles. If necessary, spin the plate briefly in a plate centrifuge to ensure all reagents are at the bottom of the wells. Label the plate, cover with a lid and store on ice or at 4 °C.

7. Set up the Sciclone deck

Confirm that the software has correctly read the workbook and is set to run the correct number of columns. If the incorrect number of columns is indicated, modify and save the KAPA Library Prep workbook as described in Step 1, then start the application again from the Main method.

Step through the pictures, placing the appropriate consumables/prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

8. Run the KAPA Library Prep Steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the library prep protocol.

The Maestro KAPA Library Prep Application will automatically proceed through End Repair, A-tailing, Ligation, and PCR setup steps as indicated in the flowchart on page 4. While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. See Appendix A for a Step-by-Step guide to the Sciclone NGS Workstation steps of the KAPA Library Prep Application.

If the “Size Selection” option has been set for the library prep workflow, the application will pause at the completion of the post-ligation SPRI cleanup. A message will prompt the user to replace the plate at C2 with a plate containing 20 µL fresh AMPureXP® beads and to replace the plate at B2 with fresh PEG/NaCl SPRI solution.

When the PCR setup is complete, the application will pause and show a message indicating that the PCR plate should be sealed and placed on a thermocycler for the amplification step. Close the dialog box to complete the run and shut down the leg lights and Inheco temperature controls.

Note: The 10 mM Tris pH 8.0 and 80% EtOH plates may be retained for use in the Post-PCR SPRI cleanup.

PCR Amplification

KAPA recommends the following program for amplification of libraries. The number of cycles should be optimized according to the amount of input DNA in the sample. Use a heated lid to prevent condensation.

PCR:

98 ˚C for 45 seconds 5-10* cycles of: 98 ˚C for 15 seconds 60 ˚C for 30 seconds 72 ˚C for 30 seconds 72 ˚C for 60 seconds Hold at 4 ˚C *Please consult the KAPA HTP NGS Library Preparation Technical Guide for information regarding optimization of library amplification.

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KAPA Post-PCR SPRI Run Preparation Steps

The KAPA Post-PCR SPRI cleanup step is provided as a separate Maestro application. If desired, the user may designate a different liquid handler for post-PCR sample processing to avoid any possible cross-contamination of pre-amplification samples. Post-PCR SPRI cleanup applications are available for both the Sciclone NGS and the Zephyr™ NGS Workstations. The instructions here are for running the KAPA Post-PCR SPRI application on the Sciclone NGS Workstation.

Note: AMPureXP® beads should be equilibrated to room temperature for about 30 minutes before use. They should be taken out of 4 °C storage when the PCR Amplification step is started.

1. Open the KAPA Library Prep Workbook

In the KAPA Post-PCR SPRI worksheet, enter the number of columns to be processed and save the workbook. The workbook must be located in the filepath: C:\ProgramData\Caliperls\Maestro\Workbooks\ and must have the name “KAPA Library Prep Workbook.xls.” If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

Figure 7. KAPA Post-PCR SPRI worksheet.

3. Prepare the AMPure® XP Beads Plate and 10 mM Tris pH 8.0 Plate

Aliquot 55 µL of AMPure XP® reagent (resuspended beads) per well in a BioRad Hardshell® PCR plate. Ensure that the beads are at room temperature and well mixed prior to pipetting.

If the 96-well BioRad Hardshell® PCR plate containing 10 mM Tris pH 8.0 used in the KAPA Library Prep application is saved at the completion of the library prep steps, the same plate may be used during the post-PCR cleanup. Confirm that enough volume remains in the wells (30 µL plus >5 µL overage volume in each well), or set up a new plate as indicated in the workbook.

4. Prepare 80% Ethanol Reservoir

If the 80% EtOH reservoir used in the KAPA Library Prep run has been retained, it can be used for this run. Otherwise, make 50 mL fresh 80% EtOH solution by diluting 40 mL 100% Ethanol with 10 mL nuclease-free molecular biology grade water. Pour 50 mL fresh 80% EtOH into a Seahorse deepwell reservoir and cover with a lid.

5. Setup the Sciclone Deck

Confirm that the software has correctly read the workbook and is set to run the correct number of columns. Step through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

Note: When the KAPA Post-PCR SPRI application is started, the variables used for tip tracking are reset. The run must be started with new, full tip boxes in the indicated positions, as Maestro will not retain tip tracking information from the library prep run.

6. Run the “KAPA Post-PCR SPRI” steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the library prep steps. See Appendix B for a Step-by-Step guide to the Sciclone steps of the KAPA Post-PCR SPRI application.

When the run is complete, the application will pause and show a message indicating that the sample plate should be sealed and stored appropriately. Close the dialog box to complete the run and shut down the leg lights and Inheco temperature controls.

2. Start the KAPA Post-PCR SPRI Application

Launch the Maestro software and open the “KAPA Post-PCR SPRI” application. Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method.

Note: When the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps.

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Library QC and storage

Seal the library plate and store at -20 °C for up to 7 days, or proceed directly into library validation prior to storing.

The LabChip® GX may be used to check the size distribution of fragments in the amplified library and estimate the concentration of fragments in the appropriate size range. Make a 1/25 dilution of library into molecular biology grade water in a BioRad Hardshell skirted 96-PCR plate. Mix well by pipetting up and down, and spin the plate to remove any bubbles. Run the samples on the GX using a High Sensitivity DNA chip and kit according to the standard LabChip protocol.

Additional/alternative validation and quantification, including qPCR quantification, should be done according to the user’s standard practices.

Figure 8: Data tracing from LabChipGX analysis of 1:25 dilutions of input DNA (red) and final library amplified with 6 PCR cycles (blue).

Figure 10: Yield data from LabChipGX analysis of 24 replicate library preps shown in figure 9.

Figure 9: Gel image from LabChipGX analysis of 1:25 dilutions of KAPA library preps from 24 replicate samples of 500 ng total Human DNA (Promega G1471).

Expected Results

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